The Ca2+-dependent secretion of catecholamines from adrenal medullary chromaffin cells is not only fundamental to the function of the adrenal medulla, but also serves as a model for exocytotic release of catechocholamines and other neurotransmitters and hormones from nerve terminals and cells. The continuing focus of the reserch is to identify and investigate the biochemical basis for exocytosis using monolayers of cultured adrenal medullary chromaffin cells. Brief incubation of chromaffin cells with low concentrations of digitonin renders the plasma membrane permeable to Ca2+, ATP and proteins and allows micromolar Ca2+ to stimulate directly exocytosis. We have recently demonstrated that not only low molecular weight species but also proteins such as trypsin (27 kDa) and myosin light chain kinase (130 kDa) can be introduced into digitonintreated cells and their effects on intracellular biochemical processes and secretion investigated. In some of the proposed studies digitomintreated cells will be used to investigate the direct effects of various manipulations on Ca2+-dependent secretion and on intracellular biochemical reactions such as protein phosphorylation (with gamma-32P)ATP as a substrate). Soluble cytosolic factors including proteins exit from digitonintreated chromaffin cells with a concomittant loss of Ca2+-dependent secretion. One important goal of the proposed studies is to determine whether Ca2+-dependent secretion can be reconstituted by adding back soluble, cytosolic constituents including proteins. Preliminary data suggest that this approach is feasible. In the proposal both intact and digitonin-permeabilized chromaffin cells will be used to investigate: 1) the role of protein kinase C in secretion; 2) ATPindependent secretion and its relationship to ATPdependent secretion; 3) mechanisms underlying the in situ osmotic stability of chromaffin granules, the secretory vesicles; 4) the role of cytosolic protein in exocytosis; 5) the role of a chromaffin granule membrane protein is exocytosis using antibodies directed against the protein; and 6) the regulation of lysosomal enyzme release and constitutive protein secretion which occur via pathways not involving chromaffin granules.